Directed Enrichment of Genomic DNA for High-Throughput Sequencing

ABSTRACT

The present invention provides microarrays of oligonucleotide primer pairs, and in particular, microarrays of primers that comprise at least one cleavable linkage. Also provided are methods to capture oligonucleotide primer pairs from one or more microarrays, and methods to use the captured oligonucleotide primer pairs, such as for amplification of a target polynucleotide sequence. In addition, methods of using a microarray to isolate, purify and/or amplify a target polynucleotide are provided.

RELATED APPLICATION

This application is a continuation of application Ser. No. 11/726,719,filed on Mar. 22, 2007, and claims the benefit of U.S. ProvisionalApplication No. 60/785,295, filed on Mar. 23, 2006. The entire teachingsof the above applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Large numbers of amplicons or polymerase chain reaction products (PCR)products are necessary to amplify every exon in a genome. For example,the amplification all of the exons in the human genome would requireapproximately 250,000 amplicons or PCR products. For each amplicon, apair of primers needs to be synthesized and an individual PCR reactionneeds to be carried out, which is a costly and unwieldy task for thetotal number of 250,000 amplicons required to sequence all of the exonsin the human genome.

Microarray technologies are available that can synthesize very smallquantities of up to about 400,000 oligonucleotides on a single solidsupport. These 400,000 oligonucleotides could potentially comprise200,000 primer pairs for an amplification reaction, such as PCR. All400,000 oligonucleotides can be liberated from the microarray into asingle (multiplex) PCR reaction using known methods. However, theresulting multiplex PCR using these 200,000 primer pairs in one reactionis likely to be uninformative due to the complexity of the reaction anddue to numerous artifacts, such as primer-dimer formation. There iscurrently no known method to segregate individual primers pairs from amicroarray into individual PCR reactions.

Thus, a need exists to segregate primer pairs from a microarray, suchthat individual primer pairs can be used, for example, in anamplification reaction.

SUMMARY OF THE INVENTION

The present invention provides microarrays of oligonucleotides, e.g.,primer pairs, and in particular, microarrays of primers that comprise atleast one cleavable linkage. Also provided are methods to captureoligonucleotide primer pairs from one or more microarrays, and methodsto use the captured oligonucleotide primer pairs, such as foramplification of a target polynucleotide sequence. In addition, methodsof using a microarray to isolate, purify and/or amplify a targetpolynucleotide are provided.

Present microarray technologies can synthesize very small quantities ofup to about 400,000 oligonucleotides on a single solid support.Therefore, these 400,000 oligonucleotides can theoretically comprisearound 200,000 primer pairs for amplifying one or more targetpolynucleotides in an amplification reaction, such as PCR. However,there is no current method to separate the 200,000 primer pairs into200,000 separate amplification reactions. Although all 400,000oligonucleotides can be liberated from the microarray into a single(multiplex) PCR reaction using known methods, the resulting multiplexPCR reaction has little informative value due to the complexity of theamplified products generated and due to numerous artifacts that resultfrom such a multiplex PCR, such as primer-dimer formation (see, e.g.,Dahl et al., Nuc. Ac. Res. (2005), 33(8): e71, and references citedtherein).

There is a need for methods to segregate primer pairs from a microarray,and methods of using such primer pairs, for example, in one or moreamplification reactions. Thus, the present invention providesmicroarrays comprising primer pairs that can be used in methods ofsegregating and/or capturing primer pairs from the microarrays such thatthe primer pairs, for example, can amplify a target polynucleotide inone or more separate amplification reactions.

The present invention provides a microarray comprising a plurality ofoligonucleotide primer pairs, wherein each oligonucleotide primer paircomprises a first oligonucleotide primer and a second oligonucleotideprimer. Each primer comprises at least one cleavable linkage forreleasing the primer from the microarray. Thus, cleaving the cleavablelinkage in each primer will release the plurality of oligonucleotideprimer pairs from the microarray.

The present invention also provides a microarray comprising a pluralityof oligonucleotides in which each oligonucleotide comprises (a) twoprimer nucleic acid sequences; (b) a first cleavable linkage which islocated at or near the microarray surface, such that cleaving the firstcleavable linkage separates each oligonucleotide from the microarraysurface; and (c) a second cleavable linkage located between the twoprimer nucleic acid sequences in each oligonucleotide, such thatcleaving the second cleavable linkage separates the two primer nucleicacid sequences. In one embodiment, the two primer nucleic acid sequencesare a primer pair.

Also provided herein is a method for producing a microarray, such that aplurality of oligonucleotide primer pairs are synthesized on themicroarray. Each oligonucleotide primer pair when synthesized is presentat a discrete location (also referred to herein as a “feature” or an“element”) on the microarray and comprises a first primer and a secondprimer. The first primer and second primer each comprise at least onecleavable linkage. The method comprises providing a microarray whichcomprises a plurality of discrete locations, and each discrete locationcomprises a first primer synthesis site and a second primer synthesissite. Each second primer synthesis site is capped with a blocking groupto prevent primer synthesis at the second primer synthesis site. Thefirst primers comprising at least one cleavable linkage are synthesizedat each first primer synthesis site on the microarray, which produces aplurality of first primers on the microarray. The method furthercomprises capping the plurality of first primers to prevent furthersynthesis of each first primer. Each second primer synthesis site isuncapped and a second primer comprising at least one cleavable linkageis synthesized at each second primer synthesis site on the microarray,which produces a plurality of second primers on the microarray. Thus,the method produces a microarray, wherein a plurality of oligonucleotideprimer pairs are synthesized on the microarray. Also provided in thepresent invention is a microarray produced by the described method.

Another aspect of the present invention is a method for capturing aplurality of oligonucleotide primer pairs on a capturing means in acapturing support, wherein each oligonucleotide primer pair is presentat a discrete location on the capturing support. In a particularembodiment, the primer pairs are located at a discrete location on amicroarray and the discrete locations of the primer pairs are maintainedwhen the primer pairs are captured on a capturing support, wherein eachdiscrete location comprises or consists essentially of one primer pair.In one embodiment, a plurality of discrete locations comprises aplurality of primer pairs, wherein each discrete location comprises orconsists essentially of one primer pair, and wherein each primer pair isthe same or different. Each oligonucleotide primer of each primer paircomprises at least one first cleavable linkage and the capturing supportcomprises a capturing means for capturing the oligonucleotide primerpairs. The method comprises capturing or transferring (e.g., embedding)each oligonucleotide primer pair present at a discrete location on amicroarray into a capturing support, capturing the oligonucleotideprimer pairs on the microarray by the capturing means and separating theoligonucleotide primer pairs from the microarray by cleaving the atleast one first cleavable linkage in each oligonucleotide primer,thereby capturing a plurality of oligonucleotide primer pairs on acapturing means in a capturing support, wherein each oligonucleotideprimer pair is present at a discrete location on the capturing support.The method can further comprise separating the capturing means from thecapturing support, thereby producing a plurality of capturing means,wherein at least a portion of the capturing means comprise anoligonucleotide primer pair.

The present invention also provides a method for capturing a pluralityof oligonucleotide primer pairs from two microarrays and the primerpairs are captured on a capturing support. Each oligonucleotide primerpair comprises a first primer that is located on a first microarray at adiscrete location and a second primer that is located on the secondmicroarray at a discrete location. Each first primer is captured from afirst microarray and each second primer is captured from a secondmicroarray. Each primer also comprises at least one cleavable linkage toseparate the primer from the microarray. The method comprises embeddinga first microarray comprising a plurality of first primers into acapturing support under conditions in which each of the first primersare captured in the capturing support and the discrete location of eachof the first primers is maintained in the capturing support. The methodfurther comprises separating the first primers from the first microarrayby cleaving the at least one cleavable linkage in the first primers andremoving the first microarray from the capturing support. Upon removalof the first microarray, the first primers remain in the discretelocations in the capturing support. In addition, the method comprisesembedding a second microarray comprising a plurality of second primersinto the capturing support under conditions in which each of the secondprimers are captured in the capturing support and the discrete locationof each of the second primers is maintained in the capturing support,and the discrete location of each of the second primers overlaps withthe discrete location of each of the first primers. When the discretelocation of the first primer and second primer overlap in the capturingsupport, each discrete location in the capturing support comprises orconsists essentially of a primer pair that can be used in anamplification reaction. The method also comprises separating the secondprimers from the second microarray by cleaving the at least onecleavable linkage in each of the second primers. Thus, the methodcaptures a plurality of oligonucleotide primer pairs on a capturingsupport, wherein each oligonucleotide primer pair comprises a firstprimer and a second primer, and wherein each first primer is capturedfrom a first microarray and each second primer is captured from a secondmicroarray.

Another aspect of the invention is a method for amplifying a targetpolynucleotide using oligonucleotide primer pairs wherein eacholigonucleotide primer pair is located at a discrete position on amicroarray. Each primer in each oligonucleotide primer pair comprise atleast one cleavable linkage. The method comprises providing a pluralityof oligonucleotide primer pairs, wherein each oligonucleotide primerpair is located at a discrete position on a microarray. A targetpolynucleotide is hybridized to at least one primer in at least oneoligonucleotide primer pair on the microarray, to produce a microarraycomprising at least one primer pair comprising at least one primerhybridized to a target polynucleotide. The microarray is embedded into acapturing support (e.g., by coating the microarray with a semisolidmatrix). The at least one primer oligonucleotide pair comprising atleast one primer hybridized to a target polynucleotide is separated fromthe microarray (e.g., liberated from the microarray surface) by cleavingthe at least one cleavable linkage in each primer to release the atleast one oligonucleotide primer pair comprising at least one primerhybridized to a target polynucleotide from the microarray, wherein theat least one oligonucleotide primer pair comprising at least one primerhybridized to a target polynucleotide is captured in the capturingsupport. The method further comprises amplifying the targetpolynucleotide under conditions in which the at least oneoligonucleotide primer pair comprising at least one primer hybridized tothe target polynucleotide amplifies the target polynucleotide bypolymerase chain reaction, such that an amplified target polynucleotideis produced.

In one embodiment, the oligonucleotide primers are synthesized on themicroarray in the conventional 3′-down orientation. In anotherembodiment, the oligonucleotide primers are synthesized in the 5′-downorientation.

In a particular embodiment, a target polynucleotide hybridized to anoligonucleotide primer on the array is copied by extension of the 3′hydroxyl of one oligonucleotide primer using DNA polymerase anddeoxynucleoside triphosphates. The hybridized target polynucleotide canbe released by chemical or thermal denaturation and discarded beforeproceeding with the embedding in a capturing support, or alternatively,the double-stranded primer extension product is cleaved after embeddingin a capturing support by treatment with a restriction endonuclease.

In a particular embodiment, each oligonucleotide primer furthercomprises at its 5′ end a restriction endonuclease recognition site fora distally cleaving restriction endonuclease. Thus, in one embodiment,the method further comprises cleaving the amplified targetpolynucleotide with a restriction endonuclease specific for therestriction endonuclease recognition site, which produces a fragment ofthe amplified target polynucleotide, such that at least a portion of theoligonucleotide primer sequence is removed. In a further embodiment, themethod further comprises joining a pair of sequencing adapters to thefragment of the amplified target polynucleotide, such that onesequencing adapter is joined to each end of the fragment, and eachsequencing adapter comprises a primer binding site. This produces anadapter-modified target polynucleotide. In a particular embodiment, theadapter-modified target polynucleotide is sequenced. In anotherparticular embodiment, the sequencing adapters that are joined to thefragment of the amplified target polynucleotide are a pair ofasymmetrical adapters. In one embodiment, the pair of sequencingadapters is a pair of asymmetrical adapters. In a particular embodiment,the pair of asymmetrical adapters comprise:

a) a first oligonucleotide adapter selected from the group consistingof:

-   -   (i) an asymmetrical tail adapter comprising a first ligatable        end, and a second end comprising a single-stranded 3′ overhang        of at least about 8 nucleotides;    -   (ii) an asymmetrical Y adapter comprising a first ligatable end,        and a second unpaired end comprising two non-complementary        strands, wherein the length of the non-complementary strands are        at least about 8 nucleotides; and    -   (iii) an asymmetrical bubble adapter comprising an unpaired        region of at least about 8 nucleotides flanked on each side by a        paired region;        and        a second oligonucleotide adapter selected from the group        consisting of:    -   (iv) an asymmetrical tail adapter comprising a first ligatable        end, and a second end comprising a single-stranded 5′ overhang        of at least about 8 nucleotides, wherein the 3′ end of the        strand that does not comprise the 5′ overhang comprises at least        one blocking group;    -   (v) an asymmetrical Y adapter comprising a first ligatable end,        and a second unpaired end comprising two non-complementary        strands, wherein the length of the non-complementary strands are        at least about 8 nucleotides;    -   (vi) and    -   (vii) an asymmetrical bubble adapter comprising an unpaired        region of at least about 8 nucleotides flanked on each side by a        paired region;        wherein the nucleic acid sequence of the first and second        asymmetrical adapters are not identical.

In an alternative embodiment, the method for amplifying a targetpolynucleotide further comprises joining a universal adapter to each endof the amplified target polynucleotide to produce a firstadapter-modified target polynucleotide. Each universal adapter comprisesa primer binding site, and the 3′ end of each universal adaptercomprises a restriction endonuclease recognition site for a distallycleaving restriction endonuclease. In one embodiment, the method furthercomprises amplifying the first adapter-modified target polynucleotide toproduce an amplified adapter-modified target polynucleotide. In afurther embodiment, the method further comprises cleaving the amplifiedadapter-modified target polynucleotide with a restriction endonucleasespecific for the restriction endonuclease recognition site, whichproduces a fragment of the amplified adapter-modified targetpolynucleotide, such that at least a portion of the universal adaptersequence is removed. In a still further embodiment, the method furthercomprises joining a pair of sequencing adapters to the fragment of theamplified adapter-modified target polynucleotide to produce a secondadapter-modified target polynucleotide, such that one sequencing adapteris joined to each end of the fragment, and each sequencing adaptercomprises a primer binding site. In a particular embodiment, the secondadapter-modified target polynucleotide is sequenced. In a furtherparticular embodiment, the sequencing adapters are a pair ofasymmetrical adapters. In one embodiment, the pair of asymmetricaladapters comprise:

a) a first oligonucleotide adapter selected from the group consistingof:

-   -   (i) an asymmetrical tail adapter comprising a first ligatable        end, and a second end comprising a single-stranded 3′ overhang        of at least about 8 nucleotides;    -   (ii) an asymmetrical Y adapter comprising a first ligatable end,        and a second unpaired end comprising two non-complementary        strands, wherein the length of the non-complementary strands are        at least about 8 nucleotides; and    -   (iii) an asymmetrical bubble adapter comprising an unpaired        region of at least about 8 nucleotides flanked on each side by a        paired region;        and        a second oligonucleotide adapter selected from the group        consisting of:    -   (iv) an asymmetrical tail adapter comprising a first ligatable        end, and a second end comprising a single-stranded 5′ overhang        of at least about 8 nucleotides, wherein the 3′ end of the        strand that does not comprise the 5′ overhang comprises at least        one blocking group;    -   (v) an asymmetrical Y adapter comprising a first ligatable end,        and a second unpaired end comprising two non-complementary        strands, wherein the length of the non-complementary strands are        at least about 8 nucleotides;    -   (vi) and    -   (vii) an asymmetrical bubble adapter comprising an unpaired        region of at least about 8 nucleotides flanked on each side by a        paired region;        wherein the nucleic acid sequence of the first and second        asymmetrical adapters are not identical.

In a further alternative embodiment of the method for amplifying atarget polynucleotide, each oligonucleotide primer further comprises arestriction endonuclease recognition site for a distally cleavingrestriction endonuclease, and at least one oligonucleotide primer in theoligonucleotide primer pair further comprises a universal primersequence. In a particular embodiment, the capturing support furthercomprises a primer that is identical to the universal primer sequence inthe at least one oligonucleotide primer. In one embodiment, the primerthat is identical to the universal primer sequence further comprises amoiety that can be co-polymerized with a polyacrylamide gel, e.g., amoiety selected from the group consisting of acrylamide, acrylic acidand vinyl. In a further embodiment, the method further comprisesamplifying the target polynucleotide under conditions in which at leastone oligonucleotide primer and the primer that is identical to theuniversal primer sequence amplifies the target polynucleotide bypolymerase chain reaction, thereby producing an amplified targetpolynucleotide. In a particular embodiment, the method further comprisescleaving the amplified target polynucleotide with a restrictionendonuclease specific for the restriction endonuclease recognition siteto produce a fragment of the amplified target polynucleotide, wherein atleast a portion of the oligonucleotide primer sequences are removed. Ina further embodiment, the method further comprises joining a pair ofsequencing adapters to the fragment of the amplified targetpolynucleotide to produce an adapter-modified target polynucleotide,such that one sequencing adapter is joined to each end of the fragment.Each sequencing adapter comprises a primer binding site. In oneembodiment, the adapter-modified target polynucleotide is sequenced. Ina particular embodiment, the pair of sequencing adapters is a pair ofasymmetrical adapters. In one embodiment, the pair of asymmetricaladapters comprise:

a) a first oligonucleotide adapter selected from the group consistingof:

-   -   (i) an asymmetrical tail adapter comprising a first ligatable        end, and a second end comprising a single-stranded 3′ overhang        of at least about 8 nucleotides;    -   (ii) an asymmetrical Y adapter comprising a first ligatable end,        and a second unpaired end comprising two non-complementary        strands, wherein the length of the non-complementary strands are        at least about 8 nucleotides; and    -   (iii) an asymmetrical bubble adapter comprising an unpaired        region of at least about 8 nucleotides flanked on each side by a        paired region;        and        a second oligonucleotide adapter selected from the group        consisting of:    -   (iv) an asymmetrical tail adapter comprising a first ligatable        end, and a second end comprising a single-stranded 5′ overhang        of at least about 8 nucleotides, wherein the 3′ end of the        strand that does not comprise the 5′ overhang comprises at least        one blocking group;    -   (v) an asymmetrical Y adapter comprising a first ligatable end,        and a second unpaired end comprising two non-complementary        strands, wherein the length of the non-complementary strands are        at least about 8 nucleotides; and    -   (vi) an asymmetrical bubble adapter comprising an unpaired        region of at least about 8 nucleotides flanked on each side by a        paired region;        wherein the nucleic acid sequence of the first and second        asymmetrical adapters are not identical.

In a still further particular embodiment, the method for amplifying atarget polynucleotide further comprises joining to the ends of theamplified target polynucleotide a pair of asymmetrical adapters. In oneembodiment, the pair of asymmetrical adapters comprise:

a) a first oligonucleotide adapter selected from the group consistingof:

-   -   (i) an asymmetrical tail adapter comprising a first ligatable        end, and a second end comprising a single-stranded 3′ overhang        of at least about 8 nucleotides;    -   (ii) an asymmetrical Y adapter comprising a first ligatable end,        and a second unpaired end comprising two non-complementary        strands, wherein the length of the non-complementary strands are        at least about 8 nucleotides; and    -   (iii) an asymmetrical bubble adapter comprising an unpaired        region of at least about 8 nucleotides flanked on each side by a        paired region;        and        a second oligonucleotide adapter selected from the group        consisting of:    -   (iv) an asymmetrical tail adapter comprising a first ligatable        end, and a second end comprising a single-stranded 5′ overhang        of at least about 8 nucleotides, wherein the 3′ end of the        strand that does not comprise the 5′ overhang comprises at least        one blocking group;    -   (v) an asymmetrical Y adapter comprising a first ligatable end,        and a second unpaired end comprising two non-complementary        strands, wherein the length of the non-complementary strands are        at least about 8 nucleotides; and    -   (vi) an asymmetrical bubble adapter comprising an unpaired        region of at least about 8 nucleotides flanked on each side by a        paired region;        wherein the first and second asymmetrical adapters further        comprise a restriction endonuclease recognition site for a        distally cleaving restriction endonuclease, and the nucleic acid        sequence of the first and second asymmetrical adapters are not        identical. Joining the pair of asymmetrical adapters to the ends        of the amplified target polynucleotide produces a first        adapter-modified target polynucleotide. In a particular        embodiment, the method further comprises amplifying the first        adapter-modified target polynucleotide to produce an amplified        adapter-modified target polynucleotide. In one embodiment, the        amplified adapter-modified target polynucleotide is cleaved with        a restriction endonuclease specific for the restriction        endonuclease recognition site to produce a fragment of the        amplified adapter-modified target polynucleotide, wherein at        least a portion of the asymmetrical adapter sequences are        removed. In a further embodiment, the method further comprises        joining a pair of sequencing adapters to the fragment of the        amplified adapter-modified target polynucleotide to produce a        second adapter-modified target polynucleotide, such that one        sequencing adapter is joined to each end of the fragment and        each sequencing adapter comprises a primer binding site. In one        embodiment, the second adapter-modified target polynucleotide is        sequenced. In a particular embodiment, the pair of sequencing        adapters is a pair of asymmetrical adapters. In one embodiment,        the pair of asymmetrical adapters comprise:

b) a first oligonucleotide adapter selected from the group consistingof:

-   -   (i) an asymmetrical tail adapter comprising a first ligatable        end, and a second end comprising a single-stranded 3′ overhang        of at least about 8 nucleotides;    -   (ii) an asymmetrical Y adapter comprising a first ligatable end,        and a second unpaired end comprising two non-complementary        strands, wherein the length of the non-complementary strands are        at least about 8 nucleotides; and    -   (iii) an asymmetrical bubble adapter comprising an unpaired        region of at least about 8 nucleotides flanked on each side by a        paired region;        and        a second oligonucleotide adapter selected from the group        consisting of:    -   (iv) an asymmetrical tail adapter comprising a first ligatable        end, and a second end comprising a single-stranded 5′ overhang        of at least about 8 nucleotides, wherein the 3′ end of the        strand that does not comprise the 5′ overhang comprises at least        one blocking group;    -   (v) an asymmetrical Y adapter comprising a first ligatable end,        and a second unpaired end comprising two non-complementary        strands, wherein the length of the non-complementary strands are        at least about 8 nucleotides; and    -   (vi) an asymmetrical bubble adapter comprising an unpaired        region of at least about 8 nucleotides flanked on each side by a        paired region;        wherein the nucleic acid sequence of the first and second        asymmetrical adapters are not identical.

In another embodiment of the invention, provided is a method foramplifying a target polynucleotide (see FIGS. 7A-7B). The methodcomprises providing an oligonucleotide primer located at a discreteposition on a microarray, wherein the oligonucleotide primer comprisesat least one cleavable linkage. A target polynucleotide is hybridized tothe oligonucleotide primer on the microarray, wherein the targetpolynucleotide comprises a universal adapter ligated to each end of thetarget polynucleotide. Each universal adapter comprises a universalprimer sequence. After hybridization, a microarray comprising anoligonucleotide primer hybridized to a target polynucleotide isproduced. The oligonucleotide primer hybridized to the targetpolynucleotide is extended under appropriate condition to produce afirst strand primer extension product, which is located at a discretelocation on the microarray because the oligonucleotide primer waslocated at the discrete location on the microarray. The method furthercomprises removing the target polynucleotide from the first strandprimer extension product, thereby leaving the first strand primerextension product located at a discrete location on the microarray. Thefirst strand primer extension product located at a discrete location onthe microarray is embedded into a capturing support, wherein thecapturing support comprises a universal primer. The universal primerbinds to a universal primer binding site located in the first strandextension product as a result of primer extension of the universaladapter sequence on the target polynucleotide. The target polynucleotideis amplified in the capturing support under conditions in which theoligonucleotide primer hybridized to the target polynucleotide and theuniversal primer amplifies the target polynucleotide by polymerase chainreaction, whereby an amplified target polynucleotide is produced, andthereby amplifying the target polynucleotide.

In one embodiment, the oligonucleotide primer on the microarray iscleaved before amplifying the target polynucleotide in the capturingsupport to release the oligonucleotide primer from the microarray beforeamplification (see FIG. 7A). In an alternative embodiment, the methodfurther comprises cleaving the oligonucleotide primer on the microarrayafter amplifying the target polynucleotide in the capturing support torelease the amplified target polynucleotide from the microarray (seeFIG. 7B). In another embodiment, the oligonucleotide primer furthercomprises a restriction endonuclease recognition site for a distallycleaving restriction endonuclease. For example, the distally cleavingrestriction endonuclease is a Type IIs or a Type III restrictionendonuclease. Thus, in one embodiment, the method comprises cleaving theoligonucleotide primer with a restriction endonuclease specific for therestriction endonuclease recognition site after amplifying the targetpolynucleotide in the capturing support. After cleavage, a fragment ofthe amplified target polynucleotide is produced. In a preferredembodiment, at least a portion of the oligonucleotide primer sequence isremoved after cleavage with a restriction endonuclease recognition sitefor a distally cleaving restriction endonuclease.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1D are schematics of a microarray surface with four examples ofoligonucleotide cleavable linkages. These cleavable linkages can becleaved under mild conditions. The position of the cleavage is indicatedby an arrow. FIG. 1A illustrates a 5′ phosphorothiolate linkage. FIG. 1Billustrates a disulfide linkage (bond). FIG. 1C illustrates a 3′phosphorothiolate linkage. FIG. 1D illustrates a deoxyuridine linkage.

FIG. 2 is a schematic representation of one embodiment of the presentinvention. A microarray of oligonucleotides with at least one cleavablelinkage and an affinity tag (e.g., biotin) is embedded (“stamped”) intoa capturing support comprising a capturing means (e.g., a reversiblepolyacrylamide gel comprising streptavidin magnetic beads). The targetpolynucleotide (e.g., genomic DNA) is added to the oligonucleotide arraythat is embedded into the capturing support, and a nucleotide sequence(e.g., an exon) of the genomic DNA is amplified in an amplificationreaction (e.g., polymerase chain reaction (PCR)). The magnetic beadscomprising the amplified target polynucleotide is released (“liberated”)from the reversible polyacrylamide (e.g., by using periodate). Onestrand of the amplified target polynucleotide is separated from themagnetic bead by disassociating (e.g., “melting” or denaturation) adouble-stranded amplified target polynucleotide using e.g., temperaturechange. The separated strand can be further amplified e.g., in anemulsion PCR reaction.

FIG. 3 is schematic representation of another embodiment of the presentinvention. In this embodiment, target polynucleotides are amplified on amicroarray (e.g., by polony PCR), and the amplified target nucleic acidsequences are joined to sequencing adapters (e.g., asymmetricaladapters) for further amplification (e.g., in an emulsion PCR).

FIG. 4 is a schematic representation of one embodiment of the inventionwherein a microarray comprises an oligonucleotide primer pair, P1 andP2. At least one primer (e.g., P1) comprises a universal nucleic acidsequence. A third primer comprises the universal nucleic acid sequence.In one embodiment, a capturing support comprises the third primer. Inthis example, the third primer can further comprise an acrylamide group(e.g., ACRYDITE™). Amplification of the target polynucleotide isperformed with the two primers from the microarray and the universalprimer.

FIG. 5 is a schematic representation of one embodiment of the invention.A target polynucleotide is amplified in an amplification reaction (e.g.,polony PCR) comprising two oligonucleotide primers from a microarray.The amplified target nucleic acid sequences are joined to a pair ofasymmetrical adapters to produce adapter-modified target nucleic acidsequences, which can be further amplified in an amplification reactioncomprising a third primer (e.g., P3) that is complementary to at least aportion of a first primer binding site in one asymmetrical adapter and aprimer (e.g., P4) that is complementary to a second primer binding siteof a first nucleic acid strand that is synthesized by the third primerin the amplification reaction.

FIG. 6 is a schematic representation of a method comprising steps Athrough F for synthesizing an oligonucleotide primer pair at a discretelocation (feature) on a microarray. The method can be performed atmultiple locations on a microarray (either simultaneously orsequentially) to produce a plurality of oligonucleotide primer pairs,wherein each primer pair is at a discrete location on the microarray.

FIGS. 7A-7B are schematic representation of embodiments of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

Amplifying all of the exons in the human genome requires 250,000amplicons or polymerase chain reaction (PCR) products. Manufacturing therequired 250,000 primer pairs is a challenging task. Microarraytechnologies offer an attractive approach for the synthesis of verysmall quantities of up to 400,000 oligonucleotides on a single solidsupport. However, liberating or releasing these 400,000 oligonucleotidesfrom the microarray for use as amplification primers (e.g., for PCR) hasnot been useful because there is no current method for segregating theappropriate primer pairs into individual amplification reactions. It isknown that all 400,000 oligonucleotides can be released into a singlePCR amplification reaction. But, the resulting multiplex PCR reactionhas little informative value due to the complexity of the amplifiedproducts generated and due to numerous artifacts that result from such amultiplex PCR, such as primer-dimer formation. Thus, there is a need formethods to segregate primer pairs from a microarray, and methods ofusing such primer pairs, for example, in one or more amplificationreactions.

The present invention provides microarrays and methods of segregatingand/or capturing oligonucleotides, and in particular, primer pairs fromthe microarrays. Such primer pairs can be used, for example, to amplifya target polynucleotide in one or more separate amplification reactions.

As used herein, an “oligonucleotide” refers to a polynucleotidesequence, such as a primer, a probe and the like. As used herein, a“primer” refers to an oligonucleotide that can hybridize (also referredto herein as “anneal”) to a template polynucleotide sequence andinitiate synthesis of a polynucleotide that is complementary to thetemplate polynucleotide sequence. A primer pair (also referred to hereinas an “oligonucleotide primer pair”) is a pair of primers that togethercan amplify a template polynucleotide sequence in an amplificationreaction. As will be understood in the art, one primer in the primerpair is complementary to the template polynucleotide sequence (e.g., oneprimer is complementary to the 3′ end of the template polynucleotidesequence) and the second primer of the primer pair is complementary tothe first strand product synthesized in an amplification reaction fromthe first primer (e.g., the second primer is complementary to the 3′ endof the first strand product synthesized from the first primer). Theappropriate length of a primer depends on the intended use of theprimer, but typically ranges from about 5 to about 100; from about 5 toabout 75; from about 5 to about 50; from about 10 to about 35; fromabout 18 to about 22 nucleotides. A primer need not be the exactcomplementary sequence of the template sequence, but it should besufficiently complementary to hybridize to the template sequence. Theprimer that is hybridized to the template sequence can initiate primerextension under appropriate conditions, as will be appreciated by aperson of skill in the art. Conditions for the primer extension activityof a wide range of polymerase enzymes are known in the art.

In one aspect of the invention, provided is a microarray comprising aplurality of oligonucleotide primer pairs, wherein each oligonucleotideprimer pair comprises a first oligonucleotide primer and a secondoligonucleotide primer. The first oligonucleotide primer and a secondoligonucleotide primer of a primer pair are located at the same discretelocation on a microarray (see, e.g., FIG. 3). Each primer comprises atleast one cleavable linkage for releasing the primer from themicroarray. A “cleavable linkage” or “scissile linkage” is a linkage inthe oligonucleotide that permits the specific cleavage of theoligonucleotide. For example, a succinate linkage could be used (see,e.g., U.S. published patent application 2006/0035218). In a preferredembodiment the cleavable linkage can be cleaved under mild,non-denaturing conditions leaving a 3′ OH or 3′ phosphate group. As willbe understood by the person of skill in the art, a 3′ phosphate groupcan be converted into a 3′ OH group with a variety of phosphatases. Inone embodiment, the cleavable linkage is selected from the groupconsisting of a 5′ phosphorothiolate linkage, a disulfide bond, a 3′phosphorothiolate linkage and a deoxyuridine.

As will be appreciated by the person of skill in the art, the conditionsunder which an oligonucleotide is cleaved depends on which cleavablelinkage is present. Oligonucleotides comprising a phosphothiolatelinkage are described in U.S. Application No. 60/694,783, filed on Jun.28, 2005, the teachings of which are incorporated herein by reference intheir entirety. A phosphorothiolate linkage (3′ or 5′) in apolynucleotide sequence can be efficiently cleaved according to methodsknown in the art (see, e.g., Vyle, et al., Biochemistry 31: 3012-3018(1992); Sontheimer, et al., Methods 18: 29-37 (1999); Mag, et al.,Nucleic Acids Res., 19(7):1437-1441 (1991)). For example, aphosphorothiolate linkage can be cleaved chemically by exposure toparticular metal agents, e.g., silver (Ag), mercury (Hg), copper (Cu),manganese (Mn), zinc (Zn) or cadmium (Cd), among others. Water-solublesalts that provide the anions of these metals are also useful. Inaddition, Iodine (I) can be used to cleave a phosphorothiolate linkage.Silver-containing salts such as silver nitrate (AgNO₃), or other saltsthat provide silver ions (Ag+), are particularly useful in the methodsof the present invention.

Suitable conditions for cleaving a phosphorothiolate linkage include,but are not limited to, incubation with Ag+ ions at a pH in the range offrom about 4.0 to about 10.0, from about 5.0 to about 9.0 or from about6.0 to about 8.0, and at a temperature in the range of from about 15° C.to about 50° C., from about 20° C. to about 45° C., from about 25° C. toabout 40° C., from about 22° C. to about 37° C., or from about 24° C. toabout 32° C. Particular suitable conditions include, for example,incubation in the presence of 50 mM AgNO₃ at about 22° C. to about 37°C. for at least about 10 minutes at a pH of about 7.0.

An oligonucleotide comprising a disulfide bond can be cleaved usingmethods that are standard in the art, e.g., using a reducing agent suchas beta mercaptoethanol, dithiothreitol, sodium 2-mercaptoethanesulfonate, or TCEP.

An oligonucleotide comprising a deoxyuridine can be cleavedenzymatically with Uracil DNA glycosylase (UDG) and an apyrimidinic (AP)lyase using methods that are standard in the art, e.g., using the USER™(Uracil-Specific Excision Reagent; New England Biolabs), and/or amixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyaseEndonuclease VIII. UDG catalyses the excision of a uracil base, formingan abasic (apyrimidinic or AP) site while leaving the phosphodiesterbackbone intact (see, e.g., Lindhal, et al., (1997) J. Biol. Chem., 252,3286-3294 and Lindhal, (1982) Annu. Rev. Biochem., 51, 61-64). The lyaseactivity of Endonuclease VIII breaks (cleaves) the phosphodiesterbackbone at the 3′ and 5′ sides of the abasic site so that base-freedeoxyribose is released (see, e.g., Melamede, et al., (1994)Biochemistry, 33, 1255-1264 and Jiang, et al., (1997) J. Biol. Chem.,272, 32230-32239).

The present invention also provides a microarray comprising a pluralityof oligonucleotides which are attached to the microarray surface. Eacholigonucleotide comprises (a) at least two primer nucleic acidsequences; (b) a first cleavable linkage which is located at or near themicroarray surface, wherein cleaving the first cleavable linkageseparates the oligonucleotide from the microarray surface; and (c) asecond cleavable linkage located between the two primer nucleic acidsequences in each oligonucleotide, wherein cleaving the second cleavablelinkage separates the two primer nucleic acid sequences in theoligonucleotide to produce an oligonucleotide primer pair. A cleavablelinkage that is located at or near the microarray surface is a linkagethat directly links the oligonucleotide to the microarray surface, andthus, is attached to the first nucleotide at the end of anoligonucleotide, or the cleavable linkage is located near the microarraysurface, and thus, is a linkage in the oligonucleotide that is close tothe end of the oligonucleotide that is attached to the microarraysurface. For example, a cleavable linkage that is located near themicroarray surface can be located within about 1 to about 15nucleotides, within about 1 to about 10 nucleotides or within about 1 toabout 5 nucleotides to the end of the oligonucleotide that is attachedto the microarray surface.

In a particular embodiment, at least one oligonucleotide primer in eacholigonucleotide primer pair comprises a target-specific nucleotidesequence. A “target-specific nucleotide sequence” is a nucleotidesequence which is complementary to the nucleotide sequence of a targetpolynucleotide. A “target polynucleotide” can be any polynucleotide, forexample, genomic DNA, one or more exons of a gene, a complementary DNA(cDNA) sequence, RNA, intron sequences and the like. In a particularembodiment, at least one oligonucleotide primer in each oligonucleotideprimer pair optionally comprises a universal nucleic acid sequence. A“universal nucleic acid sequence” (also referred to herein as auniversal primer binding site) is a sequence that is a generally knownsequence or commonly used nucleic acid sequence, as will be understoodby a person of skill in the art. For example, the universal nucleic acidsequence can be P1, P2, SP6, T7 or an M13 sequence.

In another particular embodiment, at least one oligonucleotide primer ineach oligonucleotide primer pair further comprises an affinity tag. An“affinity tag” is a tag or a label that permits the identification,capture and/or isolation of any agent that is attached to the affinitytag. Examples of an affinity tag include biotin, digoxigenin, a hapten,a ligand, a peptide and a nucleic acid. In a particular embodiment, theaffinity tag is biotin. In a particular embodiment, the affinity tag islocated at the 5′ terminus of an oligonucleotide. An affinity tag thatis located “at the 5′ terminus” of an oligonucleotide includes anaffinity tag on the first nucleotide at the 5′ end of anoligonucleotide, or the affinity tag can be on a nucleotide that is nearthe 5′ end of an oligonucleotide, e.g., within about 1 to about 15nucleotides, within about 1 to about 10 nucleotides or within about 1 toabout 5 nucleotides, from the 5′ end of an oligonucleotide.

In a particular embodiment, the oligonucleotide primer pairs on amicroarray described herein are used in an amplification reaction. Asused herein, “amplification” or an “amplification reaction” refers tomethods for amplification of a nucleic acid sequence includingpolymerase chain reaction (PCR), ligase chain reaction (LCR), rollingcircle amplification (RCA), strand displacement amplification (SDA) andmultiple displacement amplification (MDA), as will be understood by aperson of skill in the art. Such methods for amplification comprise,e.g., primers that anneal to the nucleic acid sequence to be amplified,a DNA polymerase, and nucleotides. Furthermore, amplification methods,such as PCR, can be solid-phase amplification, polony amplification,colony amplification, emulsion PCR, bead RCA, surface RCA, surface SDA,etc., as will be recognized by one of skill in the art. It will also berecognized that it is advantageous to use an amplification method thatresults in exponential amplification of free DNA molecules in solutionor tethered to a suitable matrix by only one end of the DNA molecule.Methods that rely on bridge PCR, where both PCR primers are attached toa surface (see, e.g., WO/18957 and Adessi et al., Nucleic Acids Research(2000): 28(20): E87) result in only linear amplification, which does notproduce sufficient amounts of product to support efficient libraryconstruction for subsequent sequencing. Furthermore, the products ofbridge PCR technologies are array-bound, and would have to be cleavedfrom the support as intact double stranded DNA molecules to be usefulfor subsequent sequencing. In addition, it will be recognized that it isoften advantageous to use amplification protocols that maximize thefidelity of the amplified products to be used as templates in DNAsequencing procedures. Such protocols use, for example, DNA polymeraseswith strong discrimination against misincorporation of incorrectnucleotides and/or strong 3′ exonuclease activities (also referred to asproofreading or editing activities) to remove misincorporatednucleotides during polymerization.

In a particular embodiment, a microarray of the present inventioncomprises at least about 100, 1000, 10,000, 100,000, 250,000 or 500,000distinct elements (also referred to herein as discrete locations orfeatures), wherein each element comprises one oligonucleotide primer ofa primer pair, both oligonucleotide primers of a primer pair, or aplurality of oligonucleotides. In one embodiment, each element of themicroarray has an area that is at least about 1, at least about 5, atleast about 10, at least about 16, at least about 50 or at least about100 square microns.

Also provided herein is a method for producing a microarray comprising aplurality of oligonucleotide primer pairs, wherein each oligonucleotideprimer pair is synthesized at a discrete location on the microarray andthe oligonucleotide primer pair comprises a first primer and a secondprimer. As will be understood in the art, methods for making an arrayhaving a single oligonucleotide nucleic acid sequence at a singlefeature on a microarray are known in the art (see, e.g., U.S. Pat. Nos.5,445,934, 5,744,305, and 5,677,195, which describe methods usinglight-directed spatially parallel chemical synthesis ofoligonucleotides, and Nuwaysir, et al., (Genome Research (2002) 12:1749-1755) and U.S. Pat. No. 6,375,903, which describe oligonucleotidearrays produced by maskless photolithography using a Maskless ArraySynthesizer (MAS) instrument (NimbleGen Systems, Inc.); the teachings ofall of which are herein incorporated by reference in their entirety). Ina particular embodiment of the present invention, the first primer andsecond primer each comprise at least one cleavable linkage. The methodfor producing a microarray of the present invention comprises providinga microarray which comprises a plurality of discrete locations, and eachdiscrete location comprises a first primer synthesis site and a secondprimer synthesis site. Each second primer synthesis site is capped witha blocking group to prevent primer synthesis at the second primersynthesis site and the first primers comprising at least one cleavablelinkage are synthesized at each first primer synthesis site on themicroarray, which produces a plurality of first primers on themicroarray. The plurality of first primers are capped to prevent furthersynthesis of each first primer. Each second primer synthesis site isuncapped and a second primer comprising at least one cleavable linkageis synthesized at each second primer synthesis site on the microarray,which produces a plurality of second primers on the microarray. Methodsof using multiple protecting groups are known in the art. For example,Kwiatkowski et al. (Nucleic Acids Research (1999) 27: 4710-4714)describe levulinyl-protected phosphoramidites (TEG-O-Lev) used inconcert with a methylamino-derivatized surface. In this example, thephosphoramide linkage is stable under conditions of oligonucleotidesynthesis and ammoniacal deprotection, but is cleaved by aqueous aceticacid. As a result oligonucleotides linked to the array with thetraditional base-cleavable linkage can be synthesized on an array whichalso has methylamino-derivatized surface coupled via a TEG phosphoramidelinkage. Once the first oligonucleotide primer synthesis is completed,the second oligonucleotide can be synthesized on the hydroxyl groupprotected by the TEG-O-Lev amidite. The TEG-O-Lev protected hydroxylgroup is deprotected by with hydrazine. As will be appreciated by aperson of skill in the art, other combination of hydroxyl protectinggroups can be used to achieve the desired results (e.g., see “Protectivegroups in Organic Synthesis” Greene & Wuts, (1991) 2^(nd) Ed., Wiley &Sons, NY). Thus, the present invention provides a method for producing amicroarray, wherein a plurality of oligonucleotide primer pairs aresynthesized at discrete locations on the microarray, in addition thepresent invention provides a microarray produced by the describedmethod.

Another aspect of the present invention is a method for capturing aplurality of oligonucleotide primer pairs on a capturing means in acapturing support, wherein each oligonucleotide primer pair is capturedat a discrete location on the capturing support. In one embodiment, theplurality of oligonucleotide primer pairs are captured in the capturingsupport at discrete locations that correspond to the discrete locationsof the oligonucleotide primers on a microarray. A “capturing support” isany suitable support that can capture an oligonucleotide and retain theoligonucleotide at a discrete location corresponding to the discretelocation the oligonucleotide was present at on a microarray. Forexample, a capturing support can be a semi-solid medium, e.g., apolyacrylamide gel. In a particular embodiment, the polyacrylamide gelis a reversible polyacrylamide gel. A reversible polyacrylamide gel canbe de-polymerized using a suitable reagent, e.g., by periodate cleavageof a DATD crosslinker. In a particular embodiment, the capturing supportcomprises a capturing means for capturing the oligonucleotide primerpairs. A capturing means can capture an oligonucleotide, e.g., bycapturing an affinity tag that is present on the oligonucleotide. In oneembodiment, the capturing means comprise a plurality of magnetic beads.In a particular embodiment, the magnetic beads are each at least about0.5 microns in diameter. Capturing means and methods of using same areknown in the art, e.g., U.S. Pat. No. 6,534,262, U.S. Pat. No. 5,705,628and U.S. Pat. No. 5,898,071, the teachings of which are hereinincorporated by reference in their entirety.

In another particular embodiment, the magnetic beads comprise an agentthat binds to an affinity tag present on at least one primer. Thus, inone embodiment at least one oligonucleotide primer in each primer pairfurther comprises an affinity tag. In a particular embodiment, theaffinity tag is biotin, digoxigenin, a hapten, a ligand, a peptide or anucleic acid. In another particular embodiment, the oligonucleotidecomprises biotin and the magnetic beads comprise avidin or streptavidinfor capturing an oligonucleotide comprising biotin.

The method for capturing a plurality of oligonucleotide primer pairs ona capturing means in a capturing support, wherein each oligonucleotideprimer pair is captured at a discrete location on the capturing supportcomprises embedding each oligonucleotide primer pair present at adiscrete location on a microarray into a capturing support, wherein eacholigonucleotide primer on a microarray comprises at least one firstcleavable linkage. The oligonucleotide primer pairs are captured fromthe microarray in the capturing support by the capturing means. Theoligonucleotide primer pairs are separated from the microarray bycleaving the at least one first cleavable linkage in eacholigonucleotide primer. Thus, the oligonucleotide primer pairs arecaptured in the capturing support and the discrete locations of theoligonucleotide primer pairs which were present on the microarray aremaintained in the capturing support on the capturing means. In oneembodiment, the microarray is removed from the capturing support afterembedding the microarray and cleaving the oligonucleotides.

In a further embodiment, the capturing means comprising anoligonucleotide primer pair are contacted with a target nucleic acidsequence. In one embodiment, contacting the oligonucleotide primer pairwith a target nucleic acid sequence occurs under conditions in which thetarget nucleic acid sequence hybridizes to one of more of theoligonucleotide primers. Hybridization is the annealing of complementarynucleic acid sequences under appropriate conditions, as will beappreciated by a person of skill in the art. In a particular embodiment,the method further comprises amplifying the target nucleic acid sequencethat is hybridized to at least one primer in the oligonucleotide primerpair, thereby producing an amplified target nucleic acid sequence. Inone particular embodiment, the amplification reaction is an emulsionpolymerase chain reaction. In another particular embodiment, theamplified target nucleic acid sequence is sequenced.

In another embodiment, the capturing means comprising an oligonucleotideprimer pair is isolated from the capturing support before or aftercontacting the oligonucleotide primer pair with a target nucleic acidsequence. Isolating the capturing means from the capturing support canbe achieved by any known method. For example, a capturing support thatis a reversible polyacrylamide gel can be depolymerized with periodate,thereby permitting the separation of the capturing means from thecapturing support.

In an additional embodiment, the capturing means comprising anoligonucleotide primer pair and a target nucleic acid sequence that hasbeen hybridized to at least one oligonucleotide on the capturing meansis isolated from the capturing support before amplifying the targetnucleic acid sequence.

In another particular embodiment, at least a portion of at least oneoligonucleotide primer in each oligonucleotide primer pair furthercomprises a second cleavable linkage, wherein the second cleavablelinkage is different from the first cleavable linkage. Thus, in oneembodiment, the second cleavable linkage of at least a portion of atleast one oligonucleotide primer in each oligonucleotide primer pair iscleaved, thereby producing a capturing support comprising a plurality ofoligonucleotide primer pairs, wherein at least a portion of at least oneoligonucleotide primer in each oligonucleotide primer pair is liberatedfrom the capturing means in the capturing support. Under theseconditions, at least one oligonucleotide primer in a primer pair iscaptured on a capturing means, and at least a portion of the otherprimer in the primer pair is liberated or released from the capturingmeans. In a further embodiment, at least a portion of botholigonucleotide primers in the primer pair are released from thecapturing means by cleaving a cleavable linkage in the primer. Cleavageof at least a portion of at least one primer in an oligonucleotideprimer pair promotes local amplification (e.g., in a PCR amplification)of a target nucleic acid sequence in the capturing support or in anemulsion amplification reaction. For example, by incorporating apercentage of uridine instead of a thymidine at the 5′ end of one of theoligonucleotides, the oligonucleotide can be cleaved enzymatically fromthe beads to promote local PCR. Additionally, a small proportion ofuridine/thymidine at a similar position in the other oligonucleotide ofthe oligonucleotide primer pair can also be incorporated to release aportion of that oligonucleotide as well.

In a further embodiment, at least one oligonucleotide primer in eacholigonucleotide primer pair further comprises a polymerizable group. Forexample, the polymerizable group can be ACRYDITE™, acrylamide, acrylicacid, vinyl, or other suitable unsaturated moiety that can beco-polymerized into a polyacrylamide gel. The addition of apolymerizable group to an oligonucleotide allows immobilization of theoligonucleotide in a polyacrylamide capturing support, thereby limitingdiffusion of the oligonucleotide and thus, limiting diffusion of anamplification product incorporating the oligonucleotide.

In another aspect of the invention, provided is a method for capturing aplurality of oligonucleotide primer pairs on a capturing support,wherein each oligonucleotide primer pair comprises a first primer and asecond primer, each primer comprises at least one cleavable linkage, andwherein each first primer is captured from a first microarray and eachsecond primer is captured from a second microarray. The method comprisesembedding a first microarray comprising a plurality of first primers,wherein each first primer is located on the first microarray at adiscrete location, into a capturing support under conditions in whicheach of the first primers are captured in the capturing support and thediscrete location of each of the first primers is maintained in thecapturing support. The first primers are separated from the firstmicroarray by cleaving the at least one first cleavable linkage in thefirst primers. The first microarray is removed from the capturingsupport and a second microarray comprising a plurality of secondprimers, wherein each second primer is located on the second microarrayat a discrete location, is embedded into the capturing support underconditions in which each of the second primers are captured in thecapturing support and the discrete location of each of the secondprimers is maintained in the capturing support, and wherein the discretelocation of each of the second primers overlaps with the discretelocation of each of the first primers. The second primers are separatedfrom the second microarray by cleaving the at least one second cleavablelinkage in each of the second primers, thereby capturing a plurality ofoligonucleotide primer pairs on a capturing support, wherein eacholigonucleotide primer pair comprises a first primer and a secondprimer, and wherein each first primer is captured from a firstmicroarray and each second primer is captured from a second microarray.

In one embodiment, the second microarray is removed from the capturingsupport after the second primers have been separated from the secondmicroarray. In a particular embodiment, the capturing support furthercomprises a capturing means. In a further embodiment, the method furthercomprises contacting the plurality of oligonucleotide primer pairs onthe capturing support and/or on the capturing means with a targetnucleic acid sequence, and amplifying the target nucleic acid sequencein an amplification reaction, thereby producing a plurality of amplifiedtarget nucleic acid sequences. In a particular embodiment, the amplifiedtarget nucleic acid sequences are sequenced. In another embodiment, atleast a portion of at least one oligonucleotide primer in eacholigonucleotide primer pair further comprises a third cleavable linkage,wherein the third cleavable linkage is different from the first andsecond cleavable linkage. In a particular embodiment, the method furthercomprises cleaving at least a portion of the third cleavable linkage ofat least one oligonucleotide primer in each oligonucleotide primer pair,thereby producing a capturing support comprising a plurality ofoligonucleotide primer pairs, wherein at least a portion of at least oneoligonucleotide primer in each oligonucleotide primer pair is liberatedfrom the capturing means in the capturing support. Thus, in oneembodiment, the method further comprises contacting the capturingsupport comprising a plurality of oligonucleotide primer pairs with atarget nucleic acid sequence, and amplifying said target nucleic acidsequence in an amplification reaction, thereby producing an amplifiedtarget nucleic acid sequence. In one embodiment, the amplified targetnucleic acid sequence is sequenced.

In another aspect of the invention, provided is a method for amplifyinga target polynucleotide, comprising a) providing a plurality ofoligonucleotide primer pairs, wherein each oligonucleotide primer pairis located at a discrete position on a microarray, and wherein eacholigonucleotide primer in each oligonucleotide primer pair comprises atleast one cleavable linkage; b) hybridizing a target polynucleotide toat least one primer in at least one oligonucleotide primer pair on themicroarray, thereby producing a microarray comprising at least oneprimer pair comprising at least one primer hybridized to a targetpolynucleotide; c) embedding the microarray of step b) into a capturingsupport; d) separating the at least one primer oligonucleotide paircomprising at least one primer hybridized to a target polynucleotidefrom the microarray by cleaving the at least one cleavable linkage ineach primer, thereby releasing the at least one oligonucleotide primerpair comprising at least one primer hybridized to a targetpolynucleotide from the microarray, wherein the at least oneoligonucleotide primer pair comprising at least one primer hybridized toa target polynucleotide is captured in the capturing support; and e)amplifying the target polynucleotide under conditions in which the atleast one oligonucleotide primer pair comprising at least one primerhybridized to the target polynucleotide amplifies the targetpolynucleotide by polymerase chain reaction, whereby an amplified targetpolynucleotide is produced, thereby amplifying a target polynucleotide.In one embodiment, each oligonucleotide primer further comprises at the5′ end of the oligonucleotide primer a restriction endonucleaserecognition site for a distally cleaving restriction endonuclease. Asused herein, “a distally cleaving restriction endonuclease” refers to arestriction endonuclease that recognizes a particular site within anucleic acid sequence and cleaves this nucleic acid sequence outside theregion of the recognition site (cleavage occurs at a site which isdistal or outside the site recognized by the restriction endonuclease).In one embodiment, a restriction endonuclease that cleaves a nucleicacid distally to its restriction endonuclease recognition site cleaveson one side of the restriction endonuclease recognition site (forexample, upstream or downstream of the recognition site). In anotherembodiment, restriction endonuclease that cleaves a nucleic aciddistally to its restriction endonuclease recognition site cleaves onboth sides of the restriction endonuclease recognition site (forexample, upstream and downstream of the recognition site). In anotherembodiment, the restriction endonuclease cleaves once between tworestriction endonuclease recognition sites. In one embodiment, adistally cleaving restriction endonuclease is a Type IIs or a Type IIIrestriction endonuclease. Thus, in one embodiment, the method furthercomprises cleaving the amplified target polynucleotide with arestriction endonuclease specific for the restriction endonucleaserecognition site, thereby producing a fragment of the amplified targetpolynucleotide, wherein at least a portion of the oligonucleotide primersequence is removed.

In one particular aspect of the invention, a pair of sequencing adaptersare joined to the fragment of the amplified target polynucleotide, suchthat a sequencing adapter is joined to each end of the fragment, andwherein each sequencing adapter comprises a primer binding site, therebyproducing an adapter-modified target polynucleotide. As used herein,“joining” refers to methods such as ligation, annealing or recombinationused to attach one component to another. In one embodiment, theadapter-modified target polynucleotide is sequenced. In a particularembodiment, the pair of sequencing adapters is a pair of asymmetricaladapters. Asymmetrical adapters are described in detail in U.S.application Ser. No. 11/338,620, filed on Jan. 26, 2006, the teachingsof which are incorporated herein by reference in their entirety. In oneembodiment, a pair of asymmetrical adapters comprise:

a) a first oligonucleotide adapter selected from the group consistingof:

-   -   (i) an asymmetrical tail adapter comprising a first ligatable        end, and a second end comprising a single-stranded 3′ overhang        of at least about 8 nucleotides;    -   (ii) an asymmetrical Y adapter comprising a first ligatable end,        and a second unpaired end comprising two non-complementary        strands, wherein the length of the non-complementary strands are        at least about 8 nucleotides; and    -   (iii) an asymmetrical bubble adapter comprising an unpaired        region of at least about 8 nucleotides flanked on each side by a        paired region;

and

b) a second oligonucleotide adapter selected from the group consistingof:

-   -   (i) an asymmetrical tail adapter comprising a first ligatable        end, and a second end comprising a single-stranded 5′ overhang        of at least about 8 nucleotides, wherein the 3′ end of the        strand that does not comprise the 5′ overhang comprises at least        one blocking group;    -   (ii) an asymmetrical Y adapter comprising a first ligatable end,        and a second unpaired end comprising two non-complementary        strands, wherein the length of the non-complementary strands are        at least about 8 nucleotides; and    -   (iii) an asymmetrical bubble adapter comprising an unpaired        region of at least about 8 nucleotides flanked on each side by a        paired region;

wherein the nucleic acid sequence of the first and second asymmetricaladapters are not identical.

As used herein, two (or more) asymmetrical adapters are “non-identical”or “not identical” when the asymmetrical adapters differ from each otherby at least one nucleotide in a primer binding site, by at least onenucleotide in the complementary nucleic acid sequence of a primerbinding site, and/or by the presence or absence of a blocking group. Anasymmetrical adapter comprises a primer binding site in a region ofsingle-stranded nucleic acid sequence in the asymmetrical adapter. Thetwo (or more) non-identical asymmetrical adapters can have substantialdifferences in nucleic acid sequences. For example, two asymmetricaltail adapters, asymmetrical bubble adapters or two asymmetrical Yadapters can comprise entirely different sequences (e.g., with little orno sequence identity). In a particular embodiment, the non-identicalasymmetrical adapters have little or no sequence identity in theunpaired region (e.g., the tail region, the arms of the Y region, or thebubble region). Alternatively, a pair of asymmetrical adapters are notidentical such that they differ in kind or type, e.g., the first andsecond asymmetrical adapters are not both asymmetrical tail adapters,not both asymmetrical Y adapters, or not both asymmetrical bubbleadapters. That is, a pair of asymmetrical adapters can comprise, e.g.,an asymmetrical tail adapter and a bubble adapter or Y adapter, or apair of asymmetrical adapters can comprise a bubble and a Y adapter. Ina particular embodiment, two (or more) asymmetrical adapters that arenot identical in kind or type differ from each other by at least onenucleotide in a primer binding site, by at least one nucleotide in thecomplementary nucleic acid sequence of a primer binding, and/or by thepresence or absence of a blocking group.

In an alternative aspect of the invention, a universal adapter is joinedto each end of the amplified target polynucleotide, wherein eachuniversal adapter comprises a primer binding site, and the 3′ end ofeach universal adapter comprises a restriction endonuclease recognitionsite for a distally cleaving restriction endonuclease, thereby producinga first adapter-modified target polynucleotide. As used herein, a“universal adapter” is an adapter that comprises a universal primerbinding site. As described above, A “universal primer binding site”(also referred to herein as a universal nucleic acid sequence) is asequence that is a generally known sequence or commonly used nucleicacid sequence, as will be understood by a person of skill in the art.For example, the universal nucleic acid sequence can be an M13 sequence.In one embodiment, the method further comprises amplifying the firstadapter-modified target polynucleotide, thereby producing an amplifiedadapter-modified target polynucleotide. In a particular embodiment, themethod further comprises cleaving the amplified adapter-modified targetpolynucleotide with a restriction endonuclease specific for therestriction endonuclease recognition site, thereby producing a fragmentof the amplified adapter-modified target polynucleotide, wherein atleast a portion of the universal adapter sequence is removed. In afurther particular embodiment, a pair of sequencing adapters are joinedto the fragment of the amplified adapter-modified target polynucleotide,wherein one sequencing adapter is joined to each end of the fragment,and wherein each sequencing adapter comprises a primer binding site,thereby producing a second adapter-modified target polynucleotide. Thus,in another embodiment of the invention, the method further comprisessequencing the second adapter-modified target polynucleotide. In aparticular embodiment, the pair of sequencing adapters is a pair ofasymmetrical adapters.

In a still further alternative embodiment of the method for amplifying atarget polynucleotide, each oligonucleotide primer further comprises arestriction endonuclease recognition site for a distally cleavingrestriction endonuclease, and at least one oligonucleotide primer in theoligonucleotide primer pair further comprises a universal primersequence. In a particular embodiment, the capturing support furthercomprises a primer that is identical to the universal primer sequence inthe at least one oligonucleotide primer. In one embodiment, the primerthat is identical to the universal primer sequence further comprises apolyacrylamide group. In a further embodiment, the method furthercomprises amplifying the target polynucleotide under conditions in whichat least one oligonucleotide primer and the primer that is identical tothe universal primer sequence amplifies the target polynucleotide bypolymerase chain reaction, thereby producing an amplified targetpolynucleotide. In a particular embodiment, the method further comprisescleaving the amplified target polynucleotide with a restrictionendonuclease specific for the restriction endonuclease recognition siteto produce a fragment of the amplified target polynucleotide, wherein atleast a portion of the oligonucleotide primer sequences are removed. Ina further embodiment, the method further comprises joining a pair ofsequencing adapters to the fragment of the amplified targetpolynucleotide to produce an adapter-modified target polynucleotide,such that one sequencing adapter is joined to each end of the fragment.Each sequencing adapter comprises a primer binding site. In oneembodiment, the adapter-modified target polynucleotide is sequenced. Ina particular embodiment, the pair of sequencing adapters is a pair ofasymmetrical adapters.

In a still further alternative embodiment of the invention, the methodof amplifying a target polynucleotide comprises joining to the ends ofthe amplified target polynucleotide a pair of asymmetrical adapters,wherein the first and second asymmetrical adapters further comprise arestriction endonuclease recognition site for a distally cleavingrestriction endonuclease, and wherein the nucleic acid sequence of thefirst and second asymmetrical adapters are not identical, therebyproducing a first adapter-modified target polynucleotide. In aparticular embodiment, the method further comprises amplifying the firstadapter-modified target polynucleotide, thereby producing an amplifiedadapter-modified target polynucleotide. In one embodiment, the amplifiedadapter-modified target polynucleotide is cleaved with a restrictionendonuclease specific for the restriction endonuclease recognition site,thereby producing a fragment of the amplified adapter-modified targetpolynucleotide, wherein at least a portion of the asymmetrical adaptersequences are removed. In a further embodiment, a pair of sequencingadapters are joined to the fragment of the amplified adapter-modifiedtarget polynucleotide, wherein one sequencing adapter is joined to eachend of the fragment, and wherein each sequencing adapter comprises aprimer binding site, thereby producing a second adapter-modified targetpolynucleotide. Thus, in one embodiment, the method further comprisessequencing the second adapter-modified target polynucleotide. In oneembodiment, the pair of sequencing adapters is a pair of asymmetricaladapters.

In one embodiment, the oligonucleotide primers are synthesized on themicroarray in the conventional 3′-down orientation. In anotherembodiment, the oligonucleotide primers are synthesized in the 5′-downorientation.

In a particular embodiment, a target polynucleotide hybridized to anoligonucleotide primer on the array is copied by extension of the 3′hydroxyl of one oligonucleotide primer using DNA polymerase anddeoxynucleoside triphosphates. The hybridized target polynucleotide canbe released by chemical or thermal denaturation and discarded beforeproceeding with the embedding in a capturing support, or alternatively,the double-stranded primer extension product is cleaved after embeddingin a capturing support by treatment with a restriction endonuclease.Thus, in one embodiment of the invention, only a single cleavable primersequence is required at each microarray element, and a universal adapteris used to provide the second primer nucleic acid sequence required forsubsequent PCR. In this embodiment, the oligonucleotide primers of thearray are attached to the microarray surface at their 5′ ends. Methodsto produce such arrays are well known in the art, e.g., by ink jetdeposition of oligonucleotides having a reactive group at the 5′ end orby light-directed array synthesis methods using 5′-O-phosphoramidites. Acleavable linkage is incorporated at or near the attachment site at the5′ end of each oligonucleotide primer on the microarray. The nucleicacid sequence of the oligonucleotide primers located at each element ofthe microarray is designed such that extension of the primers produces afirst strand extension product comprising the target polynucleotide. Thetarget polynucleotide can then be further characterized, e.g., by DNAsequencing. For example, genomic DNA from a sample to be analyzed can berandomly sheared to an appropriate size range (e.g., at least about 300base pairs to at least about 1000 base pairs). The fragments can beblunt-ended and ligated to an excess of universal adapters comprising auniversal primer site or universal primer binding site, therebyproducing adapter-ligated fragments. The adapter-ligated fragments canbe optionally gel-purified to produce a collection of adapter-ligatedfragments of homogeneous size range and distribution (e.g.,approximately 500 bp, ±50 bp), and/or to remove excess adapters. Theadapter-ligated fragments are hybridized to the oligonucleotide primerson the microarray, preferably under stringent hybridization conditionsand excess unhybridized fragments, or weakly hybridized fragments, areoptionally removed using high stringency wash conditions. The 3′ ends ofmicroarray oligonucleotide primers that are hybridized to acomplementary nucleic acid sequence of adapter-ligated fragments areextended, e.g., by addition of DNA polymerase and deoxynucleosidetriphosphates (dNTPs) to generate an array-bound complementary firststrand extension product of each hybridized adapter-ligated fragment.The hybridized fragments can be removed or released from the array anddiscarded. Typical conditions for removing hybridized fragments includewashing at approximately 95° C. in a stripping solution having low ionicstrength, which may also include formamide, urea, or other denaturant,or in a stripping solution having an extreme pH (above about pH 11 orbelow about pH 2.5). The array is rinsed to remove the strippingsolution, coated with a capturing support, such as a semisolid matrix(e.g., a polyacrylamide gel) to embed the array-bound first strandextension products and the microarray primers. The microarray-boundfirst strand extension products and microarray primers are cleaved toliberate (release) them from the microarray surface and into thecapturing support. Amplification, e.g., PCR, reagents including a DNApolymerase, dNTPs and a universal primer complementary to the primernucleic acid sequence in the first strand extension products (producedby extension of the oligonucleotide primer through the universal adaptersequence) are added in an appropriate buffer and amplification isperformed in each discrete location in the capturing support using theliberated microarray primers and the universal primer, resulting in aplurality of amplified target polynucleotide sequences in the capturingsupport (see FIG. 7A).

In an alternative embodiment, the microarray oligonucleotide primerscomprise a cleavable linkage but which are not cleaved and liberatedfrom the microarray immediately after embedding in the capturing support(see FIG. 7B). Amplification is carried out in the capturing support inthe presence of a universal primer. Specifically, the amplificationproducts (e.g., double-stranded amplification products) are generated indiscrete locations in the capturing support using the microarray primerand a universal primer. The amplification products are all tethered tothe microarray surface by one DNA strand via a microarray primer. Thedouble-stranded amplification products can then be cleaved and liberatedfrom the microarray by cleaving the cleavable linkage in the microarrayprimer. In one embodiment, the cleavable linkage is a restrictionendonuclease recognition site and the double-stranded PCR products arecleaved and liberated from the microarray by cleavage with a restrictionendonuclease that recognizes the restriction endonuclease recognitionsite. In a still further embodiment, the restriction endonucleaserecognition site is recognized by a distally cleaving restrictionendonuclease, wherein the distally cutting restriction endonuclease willcleave within the target polynucleotide sequence thereby liberating theamplified target polynucleotide products from the microarray andremoving the microarray primers from the target polynucleotide sequenceat the same time. In a still further embodiment the universal primeralso comprises a restriction endonuclease recognition site that isrecognized by a distally cleaving restriction endonuclease and is whichoriented in such a way that cleavage using a distally cuttingrestriction endonuclease will cleave within the target sequence justbeyond the end of the primer. This allows both primers to be removedfrom the double-stranded target sequence at the same time as they arebeing liberated from the microarray.

In an alternative embodiment, the universal primers for amplificationcomprise a polymerizable moiety (e.g., ACRYDITE™ or acrylamide) at ornear their 5′ ends. The universal primers are added prior topolymerization of the capturing support, e.g., a polyacrylamide gel,such that the universal primers are immobilized in the capturing supportafter polymerization. Amplifying a target polynucleotide with animmobilized primer produces an immobilized amplified targetpolynucleotide. In one embodiment, the universal primer comprises arecognition site for a distally cutting restriction endonuclease at ornear the 5′ end, wherein cleavage using a distally cutting restrictionendonuclease will cut within the target sequence, e.g., just beyond theend of the universal primer sequence. This permits the immobilizedamplified target polynucleotide products generated after amplificationto be cleaved and released from the capturing support for subsequentcollection and characterization, while simultaneously removing theuniversal primer sequence.

In embodiments where restriction endonuclease cleavage is used, it willbe understood by one skilled in the art that it is advantageous to treatthe starting genomic DNA with a site-specific DNA methylase that iscapable of blocking cleavage of the corresponding restrictionendonuclease cleavage sites within the target genomic sequences. A widevariety of methylases have been described, including those that blockcleavage by multiple endonucleases (e.g., CpG methylase) and those thatblock cleavage by endonucleases that cleave distally to theirrecognition sites. For example, MmeI and EcoP15I sites are not cleavedafter methylation of their recognition sites by the correspondingmethyltransferases or methyltransferase subunits (Rao et al., 2005,BBRC, 334: 803; Tucholski et al., 1998, Gene, 223: 293). By methylatingthe recognition sites in the target genomic DNA, cleavage will onlyoccur at sites within, or adjacent to the recognition sites within theprimer sequences.

In an additional aspect of the invention, the method for amplifying atarget nucleic acid sequence further comprises removing nucleic acidsequences that are not target sequences for amplification from a pool ofnucleic acid sequences (e.g., a whole genome), by hybridizing thenucleic acid sequences to a microarray. The microarray comprisesoligonucleotides that bind to nucleic acid sequences that are not targetnucleic acid sequences (e.g., repetitive DNA elements such as Aluelements). Hybridization of the pool of nucleic acid sequences to themicroarray will selectively remove those nucleic acid sequences that arenot target nucleic acid sequences for amplification. The remainingnucleic acid sequences is then enriched for target nucleic acidsequences and can be hybridized to a microarray with oligonucleotideprimer pairs specific for the target nucleic acid sequences.Amplification of the target nucleic acid sequences is performed in anyone of the methods already described. Alternatively, a microarray can beused to enrich for sequence fragments that specifically hybridize to thearray, while allowing all other (non-specific) sequences to be removedor washed away.

The relevant teachings of all the references, patents and patentapplications cited herein are incorporated herein by reference in theirentirety.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1-39. (canceled)
 40. A method for amplifying a target nucleic acid,comprising: a) providing a plurality of oligonucleotides having at leastone cleavable linkage, wherein the plurality of oligonucleotides areimmobilized to a support; b) hybridizing a target nucleic acid to atleast one immobilized oligonucleotide so as to produce atarget/oligonucleotide complex; c) embedding the target/oligonucleotidecomplex into a capturing support; d) cleaving the at least one cleavablelinkage in the immobilized oligonucleotide, so as to release the duplexinto the capturing support; and e) amplifying the target nucleic acid ofthe released duplex so as to produce an amplified target nucleic acid.41. The method of claim 40, wherein the capturing support in step (c)comprises a plurality of primer oligonucleotides which hybridize to thetarget nucleic acid.
 42. The method of claim 40, wherein the targetnucleic acid comprises a genomic DNA fragment.
 43. The method of claim40, wherein the capturing support is a polyacrylamide gel.
 44. Themethod of claim 40, wherein the polyacrylamide gel is a reversiblepolyacrylamide gel.
 45. A method for amplifying a target nucleic acid,comprising: a) providing a plurality of oligonucleotides having at leastone cleavable linkage, wherein the plurality of oligonucleotides areimmobilized to a support; b) hybridizing a target nucleic acid to atleast one immobilized oligonucleotide so as to produce atarget/oligonucleotide complex, wherein each end of the target nucleicacid is ligated to a universal adapter; c) conducting a primer extensionreaction on the target/oligonucleotide complex so as to produce a primerextension product; d) removing the target nucleic acid from the primerextension product so as to leave the primer extension productimmobilized to the support; e) embedding the immobilized primerextension product into a capturing support; and f) amplifying theimmobilized primer extension product so as to produce a plurality ofamplification products in the capturing support.
 46. The method of claim45, wherein the capturing support of step (e) comprises at least oneprimer oligonucleotide that hybridizes to the immobilized primerextension product.
 47. The method of claim 45 further comprisingcleaving the at least one cleavable linkage in the embedded immobilizedprimer extension product before step (f), so as to release the primerextension product into the capturing support.
 48. The method of claim 45further comprising cleaving the embedded immobilized primer extensionproduct after step (f) so as to release the plurality of amplificationproducts into the capturing support.
 49. The method of claim 45, whereinthe target nucleic acid comprises a genomic DNA fragment.
 50. The methodof claim 45, wherein the capturing support is a polyacrylamide gel. 51.The method of claim 45, wherein the polyacrylamide gel is a reversiblepolyacrylamide gel.
 52. A method for amplifying a target nucleic acid,comprising: a) providing a plurality of oligonucleotides having at leastone cleavable linkage, wherein the plurality of oligonucleotides areimmobilized to a support; b) hybridizing a target nucleic acid to atleast one immobilized oligonucleotide so as to produce an immobilizedtarget/oligonucleotide complex, wherein each end of the target nucleicacid is ligated to a universal adapter; c) conducting a primer extensionreaction on the target/oligonucleotide complex so as to produce animmobilized primer extension product; d) embedding the immobilizedprimer extension product into a capture support; e) cleaving the atleast one cleavable linkage so as to release the primer extensionproduct into the capturing support; f) removing the target nucleic acidfrom the primer extension product; and g) amplifying the immobilizedprimer extension product so as to produce a plurality of amplificationproducts in the capturing support.
 53. The method of claim 52, whereinthe capturing support of step (d) comprises at least one primeroligonucleotide that hybridizes to the primer extension product.
 54. Themethod of claim 52, wherein the target nucleic acid comprises a genomicDNA fragment.
 55. The method of claim 52, wherein the capturing supportis a polyacrylamide gel.
 56. The method of claim 52, wherein thepolyacrylamide gel is a reversible polyacrylamide gel.